Journal ofMedìcinalMaterials, 2022, Vol 27, Nữ (pp 72 - 76) ANTIMICROBIAL COMPOUNDS PRODUCED BY THE MARINE ACTINOMYCETE STREPTOMYCES SP G668 Do Thi Quýnh1,2, Nguyên Thuy Linh1, Le Thi Hong Minh1, Nguyên Mai Anh1, Đoan Thi Mai Huong1’2’*, Pham Van Cuong1,2’* 1Institute o f Marỉne Biochemisừy, Vietnam Academy o f Science and Technology (VAST), Hanoi, Vietnam; 2Graduate University o f Science and Technology, Vietnam Academy o f Science and Technology, Hanoi, Vietnam *Corresponding author: huongdm@imbc.vast.vn; phamvc@imbc.vast.vn (Received April 06*, 2022) Summary Antimicrobial Compounds Produced by the Marine Actinomycete Strepíomyces sp G668 From the ethyl acetate extract of the Streptomyces sp G668 strain, seven compounds were isolated: 9//-pyrido[3,4-b]inđole (1), L-tryptophan (2), indole-3-carboxyIic acid (3), 3-indolylacetic acid (4), cyclo-(Pro-Ala) (5), uracil (6), thymine (7), Their structures were elucidated by nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy (MS) and comparison with the reference data Compound exhibited moderate antibacterial activity against Escherichia coỉi ATCC25922 and compound strongly inhibited Candida aỉbicans ATCC10231 Keywords: Marine actìnomycetes, Streptomyces, Alkaloid, Indole, Cyclodipeptide Introduction antim icrobial natural Products from m arine Marine actinobacteria have been known as an important source of bioactive natural Products which possess a wide range of bioactivities such as anticancer, antiíìingal, antiviral, etc [1] Among them, Streptomyces is the largest antibiotic-producing genus in the microbial world discovered so far [2] In the framework of the project “Investigation of anti-tuberculosis and microorganisms in the South-Central region (Khanh Hoa - Binh Thuan Sea, Vietnam)”, herein we report the isolation and structural elucidation of seven compounds from the ethyl acetate extract of the Streptomyces sp G668 collected in Van Phong, Khanh Hoa province, Vietnam Their structures are described in Fig F ig l Structures of compounds 1-7 isolated from Streptomyces sp G668 M aterial and methods 2.1 General experimental procedures The ESI-MS spectra were recorded on an Agilent 1100 LC-MSD Trap spectrometer The NMR spectra were recorded on a Bruker 500.13 and 600.36 MHz spectrometer operating at 125.76 and 150.98 MHz for 13c NMR, and at 500.13 and 600.36 MHz for 'H-NMR The 'H Chemical shiíts were referenced to CDCỈ3 and CD3OD at (Sh 7.27 and 3.31 ppm, respectively, while the 13c 72 Chemical shifts were reíerenced to the Central peak at ốc 77.1 (CDCI3) and 49.0 (CD3OD) TLC (thinlayer chromatography) silica gel Merck 60 F254 wás used Column chromatography (CC) was carried out using siỉica geỉ 40-63 pm, YMC RP18 (30-50 pm) and Sephadex LH-20 2.2 Marine materials The strain G668 was isolated from the seaweed (Caulerpa serrulata (Forsk.) J Ag.) collected the 6-meter depth on the coast of Van Phong - Journal o f Medicinal Materials, 2022, Voi 27, No Khanh Hoa in May 2020 and was identiííed by Prof Do Cong Thung, Institute of Marine Environment and Resources -Vietnam Academy of Science and Technology (VAST) 2.3 Isolation, identựìcation, and ý'ermentation o f the actinomycete strain G668 The seaweed sample (0.5 g) was crushed by the glass rod in a falcon tube; 4.5 mL of sterile sea water was added The mixture was then homogenized by vortexing for minute, and the suspension was treated using a wet-heat technique (60°c for minutes) After that, 0.5 mL of the suspension was transferred to 4.5 mL sterile distilled water and vortexed for minute to aíĩord the sample solution Aliquots of 50 pL of the sample solution was spread on a petri dish of MI medium pH 7.0 (g/L) (1 g soluble starch, 0.4 g yeast extract, 0.2 g peptone, 30 g sea salt, 15 g agar) supplemented with 50 pg/mL polymycin B and cycloheximide to inhibit Gram negative bacterial and íungal contaminations After days of aerobic incubation at 28°c, the colony of the G668 actinomycete strain was transferred onto a petri dish of the medium AI (g/L) (5 g soluble starch, g yeast extract, g peptone, 30 g instaỉit ocean sea salt, 15 g agar) for the puriíícation pinpose (Fig 2) and G668 was identiTied belonging to the genus Streptomyces by using the 16S rRNA gene sequence analysis Fig Morphological appearance of G668 strain’s colonies The strain G668 was activated and inoculated into L of AI medium After days of incubation at 28°c with agitation of 150 rpm, the culture broth was used to spread on the medium surface of 50 ílasks containing 1L of rich-nutrient solid medium A1+ (g/L) (soluble starch: 10 g, yeast extract: g, peptone: g, instant ocean sea salt 30 g, mL KBr (20 mg/mL), mL FeSƠ4 (8 mg/mL), g CaCƠ3, 15 g agar) The fermentation was incubated in an incubator at 28°c and harvested on the ten days 2.4 Isoỉation o f the secondary metabolỉtes from the ethyl acetate extract o f G668 The Streptomyces sp G668 was cultured on the agar-based media for ten days and the agar was cut into small pieces and extracted with ethyl acetate (3 times X liters X 30 minute each) at 40°c The combined ethyl acetate extracts were concentrated under reduced pressure to obtain 30 g of the crude extract The ethyl acetate extract (30 g) was separated by silica gel column chromatography on a Medium-Performance Liquid Chromatography (MPLC) equipment with a gradient of CH2Cl2/MeOH (0-100% MeOH) to give 12 íractions Fl-F12 Fraction F2 (2.5 g) was hirther purified by silica gel column chromatography with CH2Cl2/acetone gradient (0-100% acetone) to give tractions F2.1-F2.8 Fraction F2.3 (42 mg) was separated by preparative TLC with nhexane/acetone (1/1) to give compound (2.0 mg) Fraction F2.5 (500 mg) was íurther puriíied by gel íiltration over Sephadex LH-20 with MeOH to give 10 íractions F2.5.1- F2.5.10 Fraction F2.5.1 (0.94 g) was washed with M-hexane/acetone to íumish compound (5 mg) Fraction F2.3 (42 mg) was separated by preparative TLC with nhexane/acetone (1/1) to give compound (2.0 mg) Fraction F2.5 (500 mg) was íurther puriííed by gel íiltration over Sephadex LH-20 with MeOH to give 10 íractions F2.5.1- F2.5.10 Fraction F2.5.3 (100 mg) was separated on silica geỉ c c , eluting with CH2Cl2/MeOH (0-30% MeOH), to give compound (3 mg) and compound (7.1 mg) Fraction F4 (1.2 g) was íurther puriíied by gel íiltration over Sephadex LH-20 (MeOH/CH2Cl2: 9/1) to give íractions F4.1F4.7 Fraction F4.3 (110 mg) was separated by silica gel column chromatography with CH2Cl2/MeOH (0-50% MeOH) to give compound (2.4 mg) Fraction F3 (3.1 g) was subjected to c c on sỉỉỉca gel with CH2CI2/MeOH (0-50% MeOH), to give íractions F3.1-F3.8 Fraction F3.5 (125 mg) was separated by Sephadex LH-20 c c using MeOH to give compound (3 mg) and (5 mg)T 9H-Pyrido[3,4-b]indole (1): White solid, mp 198-200°c, ESI-MS m/z 169 [M+H]+ 'H NMR (500 MHz, CD3OD): ỖH(ppm) 7.28 (1H, d t,J= 1.0, 8.0 Hz, H-6), 7.57 (1H, dd, i = 1.0, 8.5 Hz, H-7), 7.58 (1H, d, ý = 8.5 Hz,H-8), 8.12 (lH ,d ,J = 5.5 Hz, H-4), 8.22 (1H, d, J= 8.0 Hz, H-5), 8.31 (1H, d, J = 5.5 Hz, H-3), 8.81 (1H, s, H-1) 13C-NMR (125 MHz, CD3OD) ỏc (ppm): 112.8 (C-8), 116.1 (C-4), 120.9 (C-6), 122.2 (C-4b), 122.7 (C-5), Journal ofMedicinalMaterials, 2022, Vol 27, No 73 129.8 (C-7), 130.5 (C-4a), 134.1 (C-l), 137.8 (C9a), 138.4 (C-3), 142.8 (C-8a) L-Tryptophan (2): brown solid, mp 59-60°C, ESI-MS: m/z 205 [M+H]+, 'H-NMR (500 MHz, DMSO-ú?6): ỎH (ppm) 2.96 (1H, dd, J = 9.0, 15.0 Hz, H-8a), 3.31 (1H, dd, J = 9.0, 15.0 Hz, H-8b), 3.56 (1H, m, H-9), 6.97 (1H, dt, J= 1.0,7.5 Hz, H5) , 7.06 (1H, dt, J = 1.5, 8.0 Hz, H-6), 7.17 (1H, br.s, H-2), 7.34 (1H, d, J = 8.0 Hz, H-7), 7.60 (1H, / = 7.5 Hz, H-4), 10.74 (1H, br.s, OH) 13C-NMR (125 MHz, DMSO-í/ố): ỏc (ppm) 27.5 (C-8), 55.4 (C-9), 109.4 (C-3), 112.1 (C-7), 119.2 (C-4), 119.4 (C-5), 121.9 (C-6), 124.9 (C-2), 127.7 (C-3a), 137.0 (C-7a), 171.9 (C=0) Indole-3-carboxylic acid (3): Brown powder; mp 231-232°c, ESI-MS: m/z 162 [M+H]+, ‘HNMR (600 MHz, CD3OD): ổH (ppm) 8.08 (1H, d d , J = 7.2,1.2 Hz, H-4), 7.96 (1H, s, H-2), 7.45 (1H, d d , J= 1.2, 7.2 Hz, H-7), 7.21 (1H, d t , J = 1.2, 7.2 Hz, H-6), 7.18 (1H, d t , J= 1.2, 7.2 Hz, H-5) 13CNMR (150 MHz, CD3OD): ỏc (ppm) 168.8 (C = ), 138.2 (C-7a), 133.4 (C-2), 127.6 (C-3a), 123.6 (C6) , 122.4 (C-5), 122.0 (C-4), 112.9 (C-7), 108.9 (C-3) 3-Indolylacetic acid (4): brown solid, [a ]D 25 72° (c 0.42, CHCI3), ESÌ-MS: m/z 176 [M+H]+, 198 [M+Na]+, 'H-NMR (500 MHz, CD3OD): ỎH (ppm) 3.71 (2H, s, H-8), 6.99 (1H, t, J = 8.0 Hz, H-5), 7.09 (1H, t, J= 8.0 Hz, H-6), 7.15 (1H, s, H2), 7.32 (1H, d , J = 8.0 Hz, H-7), 7.52 (1H, d, J = 8.0 Hz, H-4) 3C-NMR (125 MHz, CD3OD): ỏc (ppm) 33.9 (C-8), 91.1 (C-3), 111.6 (C-7), 112.0 (C-4), 119.5 (C-5), 119.6 (C-6), 122.2 (C-2), 124.3 (C-7a), 128.9 (C-3a), 138.0 (C=0) Cyclo-(Pro-Ala) (5): White solid, mp 140141°c, [a]ò25 -78.2° (c 0.32, MeOH), ESI-MS: m/z 207 [M+k]+, 'H-NMR (600 MHz, CDCI3): Su (ppm) 1.48 (3H, d, J = 7.2 Hz, CH3-Í 0), 1.89 (1H, m, H-4a), 2.02 (1H, m, H-4Ị1), 2.13 (1H, m, H-5a), 2.36 (1H, m, H-5p), 3.55 (1H, m, H-3a), 3.61 (1H, m, H-3Ị3), 4.13 (2H, m, H-6, H-9) 13C-NMR (150 MHz, CDCI3): ỏc (ppm) 16.0 (CH3-IO), 22.7 (C4) , 28.1 (C-5), 45.4 (C-3), 51.2 (C-9), 59.3 (C-6), 166.3 (C-l), 170.3 (C-7) Uracil (6): White powder, mp 320-321°C; ESI-MS: m/z 113 [M+H]+, 'H NMR (500 MHz, DMSO-í/ố): ỔH (ppm) 5.44 (1H, d, J = 7.5 Hz, H5) , 7.37 (1H, d, J = 7.5 Hz, H-6) 13c NMR (125 MHz, DMSO-í/ổ): ỗc (ppm) 100.2 (C-5), 142.2 (C-6), 151.6 (C-2), 164.6 (C-4) Thymỉne (7): White powder, mp 155-156°c, 74 ESI-MS: m/z 127 [M+H]+; 'H-NMR (500 MHz, CD3OD & CDCI3) ỎH (ppm) 1.87 (3H, s, CH3), 7.15 (1H,S, H-6) 2.5 Antimicrobial assay Antimicrobial assays were canied out using Enterococcus /aecalis (ATCC299212), Staphylococcus aureus (ATCC25923), Bacỉllus cereus (ATCC14579), Escherichỉa coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), and Candida albicans (ATCC10231) Stock Solutions of samples were prepared in DMSO, and the antimicrobial assays were cairied out in 96-well microtiter plates against the microbial strains (5 X 105 CFU/mL) using a modiíication of the published method [3] After incubation for 24 hours at 37°c, the absorbance at 650 nm was measured using a microplate reader Streptomycin and cycloheximide were used as the positive reference compounds Resuỉts and dỉscussỉon Compound was isolated as white solid, mp 198-200°c The ESI-MS spectrum of showed the protonated adduct [M+H]+ at m/z 169 The ’HNMR spectrum showed the signals of an 1,2disubstituted benzene ring at